1. Field of the Invention
The invention relates to the fields of medicine, genetics, biochemistry and molecular biology. In particular, the invention relates to reagents, reagent kits, and methods for automated hybridization. More particularly, the invention relates to reagents, reagent kits, and methods for automated in situ hybridization and automated hybridization on microarrays.
2. Description of the Related Art
Nucleic acid hybridization reactions can be used to detect and characterize specific nucleotide sequences in both DNA and RNA molecules. For example, Southern blotting involves the extraction of DNA from cells or tissues and may be used to determine the genetic structure of a particular chromosome. Similarly, Northern blotting involves the extraction of RNA from cells or tissue and may be used to determine whether and how much of a particular mRNA is present in a certain tissue. Additional assay formats for detecting nucleic acids by hybridization include the following: nuclear run-on assays; slot blot assays; magnetic particle separation; reverse Northern blot assays; dot blot assays; RNase protection assays; ligase chain reaction (LCR); polymerase chain reaction (PCR); reverse transcriptase-PCR (RT-PCR); differential display RT-PCR (DDRT-PCR); in situ hybridization; and, more recently, microfabricated arrays (also referred to as “microarrays” or “gene chips”). In each of these formats, detection methods that may be employed include, among others, radioactive labels; enzyme labels; chemiluminescent labels; and fluorescent labels.
In situ hybridization is a powerful technique for, among other uses, identifying the subcellular location of nucleic acids. Since nucleic acids, no less than other macromolecules, occupy precise positions in cells and tissues, a great deal of potential information is lost when nucleic acids are extracted by homogenization. For this reason, techniques have been developed in which nucleic acid probes are used to locate specific nucleic acid sequences in situ, a procedure called in situ hybridization (ISH). ISH may be performed to analyze either DNA or RNA in cells.
In ISH analysis of DNA, labeled nucleic acid probes are hybridized to chromosomes that have been exposed briefly to very high pH or high temperature to disrupt their DNA base pairs. The chromosomal regions that bind the probe during the hybridization step are then visualized. Originally, this technique was developed using highly radioactive DNA probes, which were detected by autoradiography. The spatial resolution of the technique, however, can be greatly improved by labeling the DNA probes chemically instead of radioactively. For this purpose the probes are synthesized with special nucleotides that contain a modified side chain, and the hybridized probes are detected with an antibody (or other ligand) that specifically recognizes this side chain.
RNA in situ hybridization methods can reveal the distribution of specific RNA molecules in cells and tissues. In this case the tissues are not exposed to high pH or temperature, so the chromosomal DNA remains double-stranded and cannot bind the probe. Instead the tissue is fixed so that RNA is retained in an exposed form capable of hybridizing with a complementary DNA or RNA probe. In this way the patterns of differential gene expression can be observed in tissues.
In situ hybridization of mRNA is useful to study disease, identify potential therapeutic targets, and evaluate candidate drugs. For example, the diagnosis of breast, ovarian, and other carcinomas may be facilitated by techniques that determine the presence and expression of the c-erb2/HER-2/neu protooncogene. The c-erb2/HER-2/neu protooncogene is a member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases. Amplification and overexpression of the c-erb2/HER-2/neu protooncogene is found in about 30% of breast carcinomas and about 20% of ovarian carcinomas. Andrecheck et al. (2000) Proc. Natl. Acad. Sci. USA 97:3444.
Either DNA or RNA probes may be used for in situ hybridization. Typically, an RNA probe (“riboprobe”) is made by in vitro transcription of a cloned cDNA that encodes the gene of interest. Thus, one must have a vector containing the cDNA flanked by promoters, such as T7 and T3 promoters, in order to make a riboprobe. On the other hand, a DNA oligonucleotide probe (“oligoprobe”) may be prepared, for example, using an automated DNA synthesizer. Thus, one of skill in the art needs to know only the sequence of the gene of interest to make an oligoprobe. An additional advantage of oligoprobes for in situ hybridization is that they are more stable than riboprobes. A further advantage of oligoprobes is that, because of their short length, access and hybridization to a target may be facilitated. In addition to oligoprobes and riboprobes, DNA/RNA hybrid probes (i.e., those containing both deoxyribonucleotides and ribonucleotides) and probes containing modified nucleic acids may be used for in situ hybridization.
Instruments for the automation of in situ hybridization have recently been developed. For example, see U.S. Pat. No. 6,296,809, which is hereby incorporated by reference in its entirety. Such instruments are programmable and capable of performing in situ hybridization on multiple samples such that each sample is subject to its own staining and treatment protocol, even when each sample requires its own temperature parameters. Additionally, samples requiring de-waxing (e.g., tumor sections) can be automatically processed at the same time as other samples that do not require this preliminary step (e.g., smears). Thus, automated instruments dramatically reduce the labor and time involved in in situ hybridization, and also facilitate standardization of protocols and consistency between results.
Microarrays are arrays of many nucleic acids having different sequences, printed in specific locations in a small area on a substrate such as a glass slide. Hybridization on a microarray (“microarray hybridization”) is a powerful technique for, among other uses, simultaneously determining the expression levels of many different genes in a cell or tissue sample. For example, Schena et al. (1996) used microarrays to quantitatively monitor differential expression of heat shock and phorbol ester-regulated genes in human T cells. Schena et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93:10614. Similarly, Heller et al. (1997) demonstrated the use of gene chips to profile expression of selected human genes involved in inflammation, as well as genes expressed in peripheral human blood cells. Heller et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94:2150.
In addition, arrays of oligonucleotide probes immobilized on solid supports have been used to determine specific nucleic acid sequences in a target nucleic acid. For example, U.S. Pat. Nos. 5,202,231 and 5,002,867, as well as International Publication No. WO 93/17126, relate to the use of large numbers of oligonucleotide probes to provide the complete nucleic acid sequence of a target nucleic acid molecule.
Additional methods of using microarrays are disclosed in the following U.S. Patents, each of which is hereby incorporated by reference in its entirety. Southern (U.S. Pat. Nos. 5,700,637 and 6,054,270) discloses an apparatus and method for analyzing a polynucleotide sequence in order to perform gene polymorphism studies, genomic fingerprinting analysis, linkage analysis, mRNA characterization, gene expression studies, and sequence determinations. The polynucleotide sequence to be analyzed is labeled and applied to an array of oligonucleotides that are capable of taking part in hybridization reactions with the polynucleotide sequence. Chee (U.S. Pat. No. 5,861,242) discloses an array of oligonucleotide probes immobilized on a solid support for the analysis of a target sequence from a human immunodeficiency virus. Wang (U.S. Pat. No. 6,004,755) discloses methods for quantitative gene expression analysis in which an end-labeled target nucleic acid is contacted with an array of probe molecules stably associated with the surface of a solid support under hybridization conditions sufficient to produce a hybridization pattern. The resultant hybridization pattern is used to obtain quantitative information about the genetic profile of the end-labeled target nucleic acid sample, as well as the physiological source from which it is derived. Lockhart (U.S. Pat. No. 6,033,860) discloses probe collections immobilized on solid supports that are highly differentially expressed among developmental stages and organs. The probes can be used to prioritize potential drug targets, to monitor disease progression and remission, and to assess drug metabolism. Lockhart (U.S. Pat. No. 6,040,138) discloses methods of monitoring the expression levels of a multiplicity of genes. The methods involve hybridizing a nucleic acid sample to a high-density array of oligonucleotide probes where the high-density array contains oligonucleotide probes complementary to subsequences of target nucleic acids in the nucleic acid sample. Cronin (U.S. Pat. No. 6,045,996) discloses methods of performing nucleic acid hybridization assays on high-density substrate-bound oligonucleotide arrays, wherein the hybridization mixture includes an isostabilizing agent, a denaturing agent, or a renaturation accelerant.
There is a need in the art for reagents and methods for automated hybridization. In situ hybridization applications for use with existing automated instruments have not yet been developed. Moreover, manual manipulation of microarrays is tedious and time-consuming, and thus there is also a need for methods for automated microarray hybridization. The automation of such processes would have wide application in the medical, genetic, biochemical, and molecular biological arts. In addition, there is a need for reagents that can be used in automated in situ hybridization and automated microarray hybridization. Furthermore, there is a need for reagent kits for use in automated in situ hybridization and automated microarray hybridization.